Mycoplasma pneumoniae is the causative agent of primary atypical pneumonia and is also responsible for other respiratory syndromes such as bronchitis, bronchiolitis, pharyngitis, croup and less severe upper respiratory tract infections with the highest incidence among school children.
Current methods for the diagnosis of M. pneumoniae infection include isolation of the organisms on complex media or demonstration of seroconversion during convalescent phases of infection (Leith et al., J. Exp. Med. 157:502-514 (1983)). The mycoplasmas, such as Mycoplasma pneumoniae, are fastidious organisms, requiring complex culture media containing peptone, yeast extract, expensive animal sera, and sterol. Growth is relatively slow and reaches low cell densities compared to most bacteria. In addition, atmospheric conditions for cell growth requires the addition of carbon dioxide. For these reasons, many clinical laboratories are unable to perform culture isolation of M. pneumoniae, and consequently are left with no real ability to diagnose the presence of this important pathogenic bacterium. Given that mycoplasmas lack cell walls, antibiotics that target the bacterial cell wall, such as penicillin, have no anti-mycoplasma activity.
Consequently, it is of importance for a physician to make a diagnosis of atypical pneumonia and prescribe the appropriate antibiotic. Initiation of appropriate therapy cannot be based on culture or serology.
Detection of genomic sequences have been proposed as rapid and specific alternatives. Different PCRs for the detection of M. pneumoniae have been described, using as targets the gene coding for the P1 adhesion protein (Jensen et al., Acta Pathol. Microbiol. Immunol. Scand. 97:1046-1048 (1989); Ursi et al., Acta Pathol. Microbiol.Immunol. Scand. 100:635-639 (1992)) or the 16S rRNA gene (van Kuppeveld et al., Appl. Environ. Microbiol. 58:2606-2615 (1992)) or a DNA sequence specific for M. pneumoniae selected from a genomic library (Bernet et al., J. Clin. Microbiol., 27:2492-2496 (1989)).
Although these methods have lesser drawbacks than culturing and serology, they are still too complex to be carried out in a routine diagnostic laboratory. False negative PCR results are rather common due to inhibitors of the PCR reaction in the clinical specimen, while false-positive results may occur due to contamination of the reagents with target DNA (Razin, Mot. and Cell. Probes, 8, 497-511 (1994)).
Based on sequence divergency of the major cytadhesin gene P1 (Su et al., Infect. Immun. 58:2669-2674 (1990)), restriction enzyme fingerprinting of genomic DNA (Su et al., J. Gen. Microbiol. 137:2727-2732 (1991); Su et al, J. Clin. Microbiol. 28:1538-1540 (1990)), two-dimensional gel electrophoresis of total proteins and PCR-mediated DNA fingerprinting (Ursi et al., J. Clin. Microbiol. 32:2873-2875 (1994)), only two types are presently recognized, indicating that M. pneumoniae as a species is genetically remarkably stable.
It was suggested by Ursi and coworkers that a switch in time from one type to another could be explained by the immune status of the population against one of these two types.
Typing M. pneumoniae is of major importance because unambiguous characterization is the basis for further identification of M. pneumoniae strains. Studies based on virulence differences between one strain and the other strain could be based on type-specificity. Furthermore, a relation may exist between type and sensitivity to antibiotics like macrolides and tetracyclines. Also the spread of M. pneumoniae strains could be studied based on type differences. The prevalence of both types seems to be time and geographic dependent.
M. pneumoniae has a very small genome of approximately 720-750 kb. In Mycoplasmal 16S ribosomal RNA, there are regions with highly conserved sequences and variable regions, V1 to V9, according to the nomenclature of Neefs et al. (Nucleic Acids Res. 18 suppl:2237-2317 (1990)).
Ribosomes are of profound importance to all organisms because they serve as the only 25known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Bacterial ribosomes contain three distinct RNA molecules which, at least in Escherichia coli, are referred to as 55, 16S and 23S rRNAs. In eukaryotic organisms, there are four distinct rRNA species, generally referred to as 5S, 18S, 28S, and 5.8S. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacterium, including the mycoplasmas, and this convention will be continued herein.
An amplification system that has significant advantages over PCR amplification systems is the amplification system referred to as NASBA (nucleic acid sequence-based amplification). The NASBA methodology is disclosed in European Patent No. 0 329 822 B1. As compared to PCR, NASBA requires less user participation and fewer manipulations and steps. Another advantage is that NASBA is performed at a relatively constant temperature, ensuring that the enzymes used in the process do not lose their activity. Finally, in NASBA each cycle of the amplification process generates a plurality of RNA copies from one substrate. Therefore, it is considered preferable to use the NASBA system to amplify mycoplasmal RNA, which in turn can be detected using nucleic acid probes.
NASBA is an enzymatic process for the amplification of RNA. Four enzyme activities are required: RNA-directed DNA-polymerase, DNA-directed DNA-polymerase, RNase H and DNA-directed RNA-polymerase. The first three activities can be provided by reverse transcriptase (preferably avian myoblastosis virus reverse transcriptase (AMV-RT)), the fourth one preferably by T7 RNA-polymerase. For optimum amplification, more RNase H activity than provided by the AMV-RT can be desirable, in which case additional enzyme (e.g. E. coli RNase H) can be added to the reaction. The first step in NASBA consists of specific hybridization of a DNA primer to the RNA target followed by cDNA synthesis by RT. RNase H activity and annealing of a second primer allow synthesis of double-stranded DNA. One (or both) of the primers contain, in addition to target-specific hybridization sequences, an RNA polymerase promoter sequence (preferably for T7 RNA polymerase). Formation of a double-stranded RNA polymerase promoter suffices to initiate transcription by RNA-polymerase, resulting in multiple copies of the complementary RNA sequence (complementary to the original RNA sequence), which in turn can serve as target for a new round of NASBA amplification.
Variations in the NASBA method are considered within the scope of the present invention. For instance, one may use `destabilizing` nucleotide triphosphates in the amplification, such as ionosine triphosphate disclosed in European patent application No. 92.202.564.8, published in 1994. In addition, it is not necessary to use RNase H, as a separate enzyme, in the NASBA reaction, because it is known in the art that reverse transcriptase itself has RNase H activity under appropriate conditions, as disclosed by Sambrook et al., Molecular Cloning (1993). Other variations would be apparent to those skilled in the art.
The NASBA technique applied can be followed by a detection method like `in solution` hybridization in an enzyme-linked gel assay (ELGA) disclosed in U.S. Pat. No. 5,482,832. However, other methods can also be applied.
As in any amplification system, one must find suitable primers to amplify the sequence of interest. The need therefore exists for primer sets and hybridization probes that can be used for the amplification and subsequent detection of Mycoplasmata, particularly Mycoplasma pneumoniae.